The present invention is directed to methods for the increased production of particular fatty acids in plants. In particular, the present invention is directed to methods for increasing oleic acid in plants.
Plant oils are used in a variety of industrial and edible uses. Novel vegetable oils compositions and/or improved means to obtain oils compositions, from biosynthetic or natural plant sources, are needed. Depending upon the intended oil use, various different fatty acid compositions are desired.
For example, in some instances having an oilseed with a higher ratio of oil to seed meal would be useful to obtain a desired oil at lower cost. This would be typical of a high value oil product. In some instances, having an oilseed with a lower ratio of oil to seed meal would be useful to lower caloric content. In other uses, edible plant oils with a higher percentage of unsaturated fatty acids are desired for cardio-vascular health reasons. And alternatively, temperate substitutes for high saturate tropical oils such as palm and coconut, would also find uses in a variety of industrial and food applications.
One means postulated to obtain such oils and/or modified fatty acid compositions is through the genetic engineering of plants. However, in order to genetically engineer plants one must have in place the means to transfer genetic material to the plant in a stable and heritable manner. Additionally, one must have nucleic acid sequences capable of producing the desired phenotypic result, regulatory regions capable of directing the correct application of such sequences, and the like. Moreover, it should be appreciated that in order to produce a desired phenotype requires that the Fatty Acid Synthetase (FAS) pathway of the plant is modified to the extent that the ratios of reactants are modulated or changed.
Higher plants appear to synthesize fatty acids via a common metabolic pathway. In developing seeds, where fatty acids are attached to glycerol backbones, forming triglycerides, are stored as a source of energy for further germination, the FAS pathway is located in the proplastids. The first committed step is the formation of acetyl-ACP (acyl carrier protein) from acety-CoA and ACP catalyzed by the enzyme, acetyl-CoA:ACP transacylase (ATA). Elongation of acetyl-ACP to 16- and 18-carbon fatty acids involves the cyclical action of the following sequence of reactions: condensation with a two-carbon unit from malonyl-ACP to form a xcex2-ketoacyl-ACP (xcex2-ketoayl-ACP synthase), reduction of the keto-function to an alcohol (xcex2-ketoacyl-ACP reductase), dehydration to form an enoyl-ACP (xcex2-hydroxyacyl-ACP dehydrase), and finally reduction of the enoyl-ACP ACP to form the elongated saturated acyl-ACP (enoyl-ACP reductase). xcex2-ketoacyl-ACP synthase I, catalyzes elongation up to palmitoyl-ACP (C16:0), whereas xcex2-ketoacyl-ACP synthase II catalyzes the final elongation to stearoyl-ACP (C18:0). Common plant unsaturated fatty acids, such as oleic, linoleic and a-linolenic acids found in storage triglycerides, originate from the desaturation of stearoyl-ACP to form oleoyl-ACP (C18:1) in a reaction catalyzed by a soluble plastid xcex94-9 desaturase (also often referred to as xe2x80x9cstearoyl-ACP desaturasexe2x80x9d). Molecular oxygen is required for desaturation in which reduced ferredoxin serves as an electron co-donor. Additional desaturation is effected sequentially by the actions of membrane bound xcex94-12 desaturase and xcex94-15 desaturase. These xe2x80x9cdesaturasesxe2x80x9d thus create mono- or polyunsaturated fatty acids respectively.
A third xcex2-ketoacyl-ACP synthase has been reported in S. oleracea leaves having activity specific toward very short acyl-ACPs. This acetoacyl-ACP synthase or xe2x80x9cxcex2-ketoacyl-ACPxe2x80x9d synthase III has a preference to acetyl-CoA over acetyl-ACP, Jaworski, J. G., et al., Plant Phys. (1989) 90:41-44. It has been postulated that this enzyme may be an alternate pathway to begin FAS, instead of ATA.
Obtaining nucleic acid sequences capable of producing a phenotypic result in FAS, desaturation and/or incorporation of fatty acids into a glycerol backbone to produce an oil is subject to various obstacles including but not limited to the identification of metabolic factors of interest, choice and characterization of an enzyme source with useful kinetic properties, purification of the protein of interest to a level which will allow for its amino acid sequencing, utilizing amino acid sequence data to obtain a nucleic acid sequence capable of use as a probe to retrieve the desired DNA sequence, and the preparation of constructs, transformation and analysis of the resulting plants.
Thus, the identification of enzyme targets and useful plant sources for nucleic acid sequences of such enzyme targets capable of modifying fatty acid compositions are needed. Ideally an enzyme target will be amenable to one or more applications alone or in combination with other nucleic acid sequences, relating to increased/decreased oil production, the ratio of saturated to unsaturated fatty acids in the fatty acid pool, and/or to novel oils compositions as a result of the modifications to the fatty acid pool. Once enzyme target(s) are identified and qualified, quantities of purified protein and purification protocols are needed for sequencing. Ultimately, useful nucleic acid constructs having the necessary elements to provide a phenotypic modification and plants containing such constructs are needed.
The present invention is directed to methods for producing plant oils having elevated levels of oleic acid (C18:1) as a percentage of the total fatty acids.
The method generally comprises growing a plant-containing a construct having as operably linked components in the 5xe2x80x2 to 3xe2x80x2 direction of transcription, a promoter region functional in a host plant cell, at least a portion of a nucleic acid sequence encoding a xcex2-ketoacyl ACP synthase in an antisense orientation and a transcription termination sequence.
The methods described herein are utilized to produce plants with increased levels of oleic acid. Increases of at least 5 percent to 60 percent, preferably, 10 percent to 50 percent, more preferably 10 percent to 40 percent over the wild type seed oil are encompassed by the methods provided herein.
In one embodiment of the present invention, a Brassica seed oil having increased oleic acid is obtained using the methods of the present invention. The oleate content of the Brassica seed oil preferably comprises greater than 65%, more preferably greater than about 75% of the fatty acid moieties in the oil. The oil of the present invention may be used as a blending source to make a blended oil product, or it may also be used in the preparation of food.
In another embodiment of the present invention, a Brassica oil having a decreased polyunsaturated fatty acid composition is obtained using the methods described herein. Brassica oils with polyunsaturated fatty acid compositions of less than about 12 weight percent are exemplified herein.